The goal is to identify and define the various regulatory processes which are involved in the induction, biosynthesis and secretion of factor D as well as mechanisms for its enzymatic activation and regulation. Factor D is a trace serum protease which is absolutely essential for activation of the alternative pathway of complement and has been hypothesized to exist in serum only as the active enzyme. Despite its key role in the formation of the C3 convertase, very little information is currently available on its cellular expression and regulation. Very recent structural analysis of a cDNA clone for factor D in our laboratory has demonstrated that the protein must be synthesized as a zymogen. This project will attempt to extend these studies and focus attention on elucidation of the key steps which are important for activation of the zymogen and for control of its activity in serum. The specific objectives are: 1) to clone and sequence full-length cDNA for factor D, 2) to isolate and examine the organization of the factor D genome and identify potential regulatory sites in the gene structure which may be important in its expression, 3) to map the chromosomal location of the factor D gene using human-rodent somatic-cell hybrids, 4) to search for evidence of restriction fragment length polymorphisms, 5) to prepare monosppecific, polyclonal antisera using synthetic peptides corresponding to specific deduced sequences in the zymogen, 6) to investigate the induction, synthesis, secretion and mechanism of activation of the zymogen, 7) to examine the possibility that natural inhibitor(s) of factor D activity are present in serum, 8) to characterize the putative protease which is hypothesized to convert the proenzyme of factor D to the active form found in the factor D convertase, 10) to investigate the potential of using expression-vector systems in order to produce large-scale amounts of the precursor. Together such studies will provide additional insight into the regulation and expression of factor D and should contribute to a better understanding of how the early events in the activation of the alternative pathway of complement function in both host defense reactions and in the pathogenesis of human inflammatory diseases.